RESUMEN
We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.
Asunto(s)
Calcio , Colorantes , Animales , Calcio/metabolismo , Color , Colorantes Fluorescentes , Indicadores y Reactivos , Microscopía Fluorescente/métodos , Ratas , RodaminasRESUMEN
Fluorescence nanoscopy methods based on the RESOLFT principle, such as beam-scanning STED nanoscopy, require the co-alignment of optical beams for molecular state (on/off) switching and fluorescence excitation. The complexity and stability of the beam alignment can be drastically simplified and improved by using a single-mode fibre as the sole light source for all required laser beams. This in turn then requires a chromatic optical element for shaping the off-switching beam into a focal-plane donut while simultaneously leaving the focal intensity distributions at other wavelengths shaped as regular focal spots. Here we describe novel designs of such so-called 'easySTED phase plates' and provide a rationale how to find the desired spectral signature for combinations of multiple wavelengths.
Asunto(s)
Luz , Microscopía Fluorescente/métodosRESUMEN
Short linear motifs (SLiMs) are protein binding modules that play major roles in almost all cellular processes. SLiMs are short, often highly degenerate, difficult to characterize and hard to detect. The eukaryotic linear motif (ELM) resource (elm.eu.org) is dedicated to SLiMs, consisting of a manually curated database of over 275 motif classes and over 3000 motif instances, and a pipeline to discover candidate SLiMs in protein sequences. For 15 years, ELM has been one of the major resources for motif research. In this database update, we present the latest additions to the database including 32 new motif classes, and new features including Uniprot and Reactome integration. Finally, to help provide cellular context, we present some biological insights about SLiMs in the cell cycle, as targets for bacterial pathogenicity and their functionality in the human kinome.
Asunto(s)
Bases de Datos de Proteínas , Células Eucariotas/metabolismo , Interacciones Huésped-Patógeno/genética , Anotación de Secuencia Molecular , Proteínas/química , Programas Informáticos , Secuencias de Aminoácidos , Animales , Bacterias/genética , Bacterias/metabolismo , Sitios de Unión , Ciclo Celular/genética , Células Eucariotas/citología , Células Eucariotas/microbiología , Células Eucariotas/virología , Hongos/genética , Hongos/metabolismo , Humanos , Internet , Modelos Moleculares , Plantas/genética , Plantas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , Virus/genética , Virus/metabolismoRESUMEN
Neurofilaments are one of the main cytoskeletal components in neurons; they can be found in the form of oligomers at pre- and postsynapses. How their presence is regulated at the postsynapse remains largely unclear. Here we systematically quantified, by immunolabeling, the occurrence of the neurofilament isoform triplet neurofilament light (NFL), medium (NFM), and heavy (NFH) at the postsynapse using STED nanoscopy together with markers of synaptic strength and activity. Our data show that, within dendritic spines, neurofilament isoforms rarely colocalize with each other and that they are present to different extents, with NFL being the most abundant isoform. The amount of the three isoforms correlates with markers of postsynaptic strength and presynaptic activity to varying degrees: NFL shows the highest correlation to both synaptic traits, suggesting its involvement in synaptic response, while NFM exhibits the lowest correlations. By quantifying the presence of neurofilaments at the postsynapse within the context of the synaptic status, this work sheds new light on the regulation of synaptic neurofilaments and their possible contribution to synaptopathies.
Asunto(s)
Filamentos Intermedios , Proteínas de Neurofilamentos , Espinas Dendríticas , Neuronas , SinapsisRESUMEN
Neurons are highly asymmetric cells that span long distances and need to react promptly to local demands. Consequently, neuronal secretory pathway elements are distributed throughout neurites, specifically in post-synaptic compartments, to enable local protein synthesis and delivery. Whether and how changes in local synaptic activity correlate to post-synaptic secretory elements is still unclear. To assess this, we used STED nanoscopy and automated quantitative image analysis of post-synaptic markers of the endoplasmic reticulum, ER-Golgi intermediate compartment, trans-Golgi network, and spine apparatus. We found that the distribution of these proteins was dependent on pre-synaptic activity, measured as the amount of recycling vesicles. Moreover, their abundance correlated to both pre- and post-synaptic markers of synaptic strength. Overall, the results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas.
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Neuronas/metabolismo , Vías Secretoras/fisiología , Membranas Sinápticas/metabolismo , Animales , Dendritas/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Aparato de Golgi/metabolismo , Masculino , Microscopía Confocal/métodos , Neuronas/fisiología , Cultivo Primario de Células , Ratas , Ratas Wistar , Sinapsis/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Mutations in the tumor suppressor p53 are among the most highly occurring events in colorectal cancer (CRC). Such mutations have been shown to influence the sensitivity of cancer cells to chemotherapeutic agents. However their impact on the efficacy of the proteasomal inhibitor bortezomib remains controversial. We thus re-evaluated the toxicity of bortezomib in the CRC cell lines HCT116 wt (wild-type) and its p53-/- clone. Transient resistance to bortezomib treatment was observed in p53-null cells that was later accompanied by an increase in levels and nuclear translocation of TAp73, an isoform of the p53-homologue p73, as well as induction of apoptosis. Knockdown of p73 in p53-/- cells using CRISPR/Cas9 significantly prolonged the duration of resistance. Moreover, similar results were observed in HT-29 cells carrying mutated p53, but not human fibroblasts with expression of functional p53. Thus, our results clearly demonstrated that TAp73 served as a substitute for p53 in bortezomib-induced apoptosis in p53-deficient or mutated cells, implicating that TAp73 could be a potential therapeutic target for treatment of CRCs, in particular those lacking functional p53.
Asunto(s)
Apoptosis/efectos de los fármacos , Bortezomib/farmacología , Mutación , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genética , Antineoplásicos/farmacología , Sistemas CRISPR-Cas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HT29 , Humanos , Factores de Tiempo , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
BACKGROUND: The maintenance or expansion of regulatory T (Treg) cells has a fundamental role in the achievement of immunological tolerance after transplantation. Here we aimed to determine mechanisms of human Treg cell depletion and reconstitution after anti-CD25 monoclonal antibody (mAb) treatment. METHODS: Seventeen patients with type 1 diabetes who received pancreatic islet transplantation and anti-CD25 mAb as induction therapy were studied. RESULTS: We observed an almost complete depletion of Treg cells after injection of anti-CD25 mAb. The kinetic of Treg cell depletion did not parallel the disappearance of CD25+ T cells as CD25 is also rapidly downregulated and internalized. Regulatory T cell reconstitution is completed within 6 months posttransplantation and appeared to be driven by IL-7-mediated homeostatic T cell proliferation. Anti-CD25 mAb treatment sensitizes Treg cell to the biological effect of IL-7, possibly rendering more common γc-chain available to interact with CD127. Homeostatic Treg cell proliferation is resistant to the inhibitory effect of rapamycin and FK506 but can be blocked by the presence of mycophenolate mofetil. CONCLUSIONS: Our data suggest that a compensatory mechanism of IL-7-mediated homeostatic proliferation can restore the inhibitory network of Treg cell after anti-CD25 induction therapy in islet allotransplantation.